Purpose: :
Our long-term goal is to develop inducible gene therapy vectorswhich will express a transgene under glaucomatous conditions.Our purpose here was to identify promoter sequences which wouldrespond to elevated IOP and drive up the expression of a reportergene. We selected the Matrix Gla (MGP) gene, whose expressionwe have shown to be highly upregulated by elevated IOP (HP)in the human trabecular meshwork (TM) tissue (1).

Methods: :
The MGP DNA promoter sequences (-748/+30) were amplified frompooled human genomic DNA (gDNA), sequence-verified and insertedinto a promoterless secreted alkaline phosphatase (SEAP) reportervector. The expression cassette (MGPpromoter-SEAP) was clonedinto a pShuttle vector to generate a replication deficient Adenovirus(AdhMGP.SEAP). Human perfused anterior segments (n=2 pairs atsubmission) were perfused at 3-4 µl/min for 24h and thenvalve-injected with 20µl of the AdhMGP.SEAP high-titerstock. After infection, the flow of one eye was raised to achievea ΔP of 30 mmHg, while the contralateral eye was left at baselinepressure (CP). Eyes were further perfused at their new constantP for 4 days (OD ΔP=30; OS ΔP=0). Effluents were collected everyday for analysis of SEAP (chemiluminescence assay). At the endof the experiment, TM tissues were dissected and halved forRNA or DNA extraction to quantify SEAP transcripts and viralgenomes by TaqMan PCR. Amplification values were normalizedby 18S.cDNA and 18S.gDNA respectively. SEAP levels were normalizedby the number of viral genomes in each TM.

Results: :
SEAP was detected in all effluents at 48h and increased thereafterat each time point (2-20µg/ml), indicating that MGP sequences-748/+30 contain a strong promoter for expression in the humanTM tissue. After correction for the number of viral genomesin each TM, the SEAP levels in the effluents from the eyes subjectedto HP were 27.8X (pair #1) and 3.3X (pair #2) higher than inthe CP control eye. Similarly, the SEAP transcripts presentin the TM tissue at the end of experiment were 1.6X higher inthe HP eye than in the CP control eye (both pairs).

Conclusions: :
In addition to containing promoter signaling sequences, theMGP -748/30 upstream-region seems to include regulatory elementsthat increase expression of the driven gene in the presenceof HP. These pressure-responding regulatory elements functionin the intact human perfused TM. Fusion of these sequences tocandidate genes would allow controlled expression of therapeuticgenes during a HP episode. This advance could help manage afuture treatment of glaucoma by gene therapy.(1) Comes and Borras.Physiol Genomics 38,205(2009)